CN112472788A

CN112472788A - Application of Furin inhibitor in preparation of medicine for treating osteoporosis

Info

Publication number: CN112472788A
Application number: CN202011400716.2A
Authority: CN
Inventors: 宋方茗; 鲜艳思; 冯晓靓; 赵劲民; 刘倩; 欧阳轶强; 梁嘉敏; 林茜茜; 陈蔚蔚; 苏元港; 韦桂宁
Original assignee: Guangxi Medical University
Current assignee: Guangxi Medical University
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2021-03-12

Abstract

The invention belongs to the technical application field of bone disease treatment, and relates to application of a Furin inhibitor in preparation of a medicine for treating osteoporosis. The invention discloses an application of CMK in preparing a medicine for treating osteoporosis, wherein the CMK has an obvious effect of resisting osteoporosis, has no toxic or side effect, is high in safety and avoids the defect of great side effect of chemical synthetic medicines. Therefore, CMK has the prospect of being developed into an anti-osteoporosis medicine and provides an effective way for treating osteoporosis.

Description

Application of Furin inhibitor in preparation of medicine for treating osteoporosis

Technical Field

The invention belongs to the technical application field of bone disease treatment, and relates to application of a Furin inhibitor in preparation of a medicine for treating osteoporosis.

Background

During the development and growth of life, bone as a highly active organ continuously undergoes two events of bone resorption and bone formation which are independent and coupled with each other, and the dynamic balance between the two events maintains the structure and function of the bone, wherein osteoblasts, which are cells that exert bone formation functions, are mainly responsible for the synthesis, secretion and mineralization of bone matrix, and osteoclasts, which are main participants of bone resorption, mediate the occurrence of diseases associated with normal physiological conditions and individual bone resorption, both of which play an important role in bone remodeling. Under normal physiological conditions, bone resorption by osteoclasts is coupled with bone formation by osteoblasts to achieve dynamic equilibrium, and the integrity and mechanical strength of bone tissues are maintained. However, with the increasing aging, the differentiation and proliferation capacity and biological functions of osteoblasts are gradually reduced, and particularly, the bone formation function of the elderly is obviously reduced, so that the bone reconstruction activity is unbalanced, the bone mass is lost, and finally, osteoporosis is caused. Currently, osteoporosis is now a worldwide public health problem. According to the International Osteoporosis group (International osteoporotosis Foundation), nearly 2 million people worldwide are reported to suffer from Osteoporosis, and more than 890 million people are fractured due to Osteoporosis every year. The anti-osteoporosis drugs commonly used in clinic at present have side effects of different degrees, and the targeting property is not obvious. Therefore, the method finds out and deeply analyzes and recognizes the differentiation and bone formation functions of osteoblasts, finds out effective key regulatory factors, is helpful for better understanding the pathogenesis of osteoporosis, finds out specific disease diagnosis indexes and develops new treatment, and further has great significance for preventing and treating osteoporosis.

Furin is a serine protease belonging to the family of precursor protein processing enzymes, and is expressed in most cells of all vertebrates and many invertebrates. It is a type I transmembrane protein consisting of 794 amino acids, and the structure comprises a signal peptide, a precursor peptide, a catalytic domain and a P domain. The widely expressed Furin is involved in the maturation process of a plurality of proteins and polypeptides in vivo, can cut various secretory precursor proteins containing specific sequences Arg-Xaa- (Lys/Arg) -Arg, comprises most peptide hormones and neuropeptides, also comprises a plurality of growth factors, receptors, matrix metalloproteases and the like, and further is involved in the physiological process of various large systems and tissues of the body. Although Furin is involved in many important physiological processes of mammals, no relevant reports are found at home and abroad in the bone development process, particularly in the differentiation of osteoblasts and the bone formation process.

Disclosure of Invention

The invention aims to develop the medical application of the Furin inhibitor and provide a reliable and effective medicament for treating osteoporosis for clinic.

The inventor obtains a conditional Furin gene knockout mouse in mesenchymal stem cells of bone marrow by pairing C57BL/6Furin-flox with Prx.1-cre mice. The results show that compared with wild mice, the bone trabecular density of mice with conditional Furin knockout is obviously increased, the number of the bone trabeculae is obviously increased, and the bone trabecular interval is obviously reduced. Suggesting that Furin may have potential targets for treating osteoporosis by affecting osteoblasts. On the basis, Furin is used as a drug target for screening drugs for treating osteoporosis, and Dec-RVKR-CMK (CMK; C) is further researched₃₄H₆₆CIN₁₁O₅) The compound is used as a specific inhibitor of Furin and has a therapeutic effect on bone loss caused by osteoporosis of ovariectomized mice. The discovery of the anti-osteoporosis effect of Dec-RVKR-CMK (CMK) proves a new medical application of CMK, namely the application in the aspect of preparing the medicine for treating osteoporosis.

The purpose of the invention is realized by the following technical scheme:

provides the application of the Furin inhibitor in preparing medicaments for treating diseases, such as osteoporosis.

Further, the Furin inhibitor is CMK.

Further, the osteoporosis is caused by ovariectomy, but is not always limited thereto.

Furthermore, the medicine for treating osteoporosis consists of CMK active components and pharmaceutically acceptable auxiliary materials.

CMK has remarkable effect of resisting osteoporosis, has no toxic and side effects, is high in safety and has specific treatment property. Therefore, CMK has the prospect of being developed into an anti-osteoporosis medicine and provides an effective way for treating osteoporosis.

Drawings

FIG. 1 is a schematic diagram of the construction of a conditional knockout mouse, wherein FIG. 1-a is a conditional knockout Furin mouse model construction strategy; FIG. 1-b shows the results of genotyping; FIG. 1-c shows the results of relative expression of mRNA; FIG. 1-d shows the results of protein expression;

FIG. 2 is a graph of the results of a Micro-CT scan of bone tissue from a wild-type, homozygous female or male mouse, wherein FIG. 2-A is a schematic bone morphology; FIG. 2-B, FIG. 2-C, FIG. 2-D, and FIG. 2-E are the statistics of the quantitative analysis of trabecular bone volume (BV/TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Tn), trabecular bone separation (Tb.Sp), respectively;

FIG. 3 is a graph of the results of an X-ray scan of the lower limb bones and the spinal bones of a wild-type, homozygous female or male mouse;

FIG. 4 is a graph showing the result of H & E, TRAcP staining in wild-type, homozygous female or male mice;

FIG. 5 is a schematic representation of the results of hard tissue sections from wild type, homozygous female or male mice;

FIG. 6 is a schematic diagram showing the results of Micro-CT scanning of a de-ovariectomized mouse model for osteoporosis;

FIG. 7 is a graph showing the results of H & E staining and TRAcP staining in a de-ovariectomized mouse model of osteoporosis.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the embodiments of the present invention are not limited to the scope of the examples. These examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. In addition, various modifications may occur to those skilled in the art upon reading the present disclosure, and such equivalent variations are within the scope of the present invention as defined in the appended claims.

Example 1

Materials and reagents

1. CMK was purchased from Cyman chemical, Inc. under lot number 14965; surgical scissors; fixing liquid;

second, Experimental methods

1. Construction of conditional knockout mice

Pairing a Furin-Flox mouse and a Prx.1-Cre mouse, carrying out gene identification on offspring to obtain heterozygote, further purifying, and finally obtaining a homozygote mouse with a condition knockout Furin gene of mesenchymal stem cells, wherein the homozygote mouse is named as Furin fl/fl, Prx.1-Cre, and a litter control mouse is wild-type Furin fl/fl; and verifying the protein level and the mRNA level of the mouse gene knockout efficiency. On the basis of the work, the size and weight changes of the wild type mice and the homozygote mice are compared by taking 12-week mice and different sexes;

scanning bone tissues of wild type and homozygote mice by using Micro-CT and X-ray, and comparing and analyzing changes of parameters such as bone morphology, bone density and the like of the mice;

hard tissue sections and H & E, TRAcP staining mice were histomorphometrically analyzed and compared to bone histomorphometric changes in wild-type, homozygous mice.

2. Ovariectomy-induced osteoporosis in mice model establishment and analysis

The establishment of the ovariectomy-induced osteoporosis mouse model was performed according to the guidelines of the animal ethics committee of the university of medical science, Guangxi. First 40 12 week old C57BL/6J mice were randomized into 4 groups: sham (PBS injection), surgery (OVX + PBS), low dose (bilateral ovariectomy + CMK 1mg/kg) and high dose (bilateral ovariectomy + CMK 10 mg/kg).

The specific modeling process is as follows, after the mouse is anesthetized by chloral hydrate, the mouse enters the abdominal cavity through a bilateral lumbar and dorsal surgical incision, and the bilateral ovaries are excised. The sham group did not remove the ovaries after finding the ovaries, and a small amount of fat was removed nearby, as in the other groups. The surgical procedure was performed under strict aseptic conditions, and the mice were placed in cages for free movement and fasting after surgery. The CMK injections were given for treatment at 1mg/kg and 10mg/kg for 1/2 days for 6 weeks post-operatively. The same amount of PBS was administered to the surgical group and the sham surgical group. After the administration is finished, animals are anesthetized by gas, eyeball blood is taken, cervical vertebra dislocation and death are carried out, then the shin bones at the two sides are separated, and the shin bone at the left side is scanned by Micro-CT to carry out scanning and 3-dimensional reconstruction analysis.

The change of bone microstructure was analyzed by histomorphometric methods and the number of osteoclasts in bone tissue after drug action was analyzed by comparison. Removing tibia of mouse, removing soft tissue, fixing in 4% paraformaldehyde for 24-48 hr, soaking tissue in 10% EDTA, and decalcifying in decalcifying machine for 5-7 days. After decalcification was completed, embedding was carried out, and H & E staining and TRAcP staining were carried out after sectioning.

2. Statistics of

All data are expressed as mean ± Standard Deviation (SD), and each experiment was repeated three times, with results expressed as the mean of three times. Data analysis was performed using SPSS 17.0 software (IBM, USA). Differences between experimental and control groups were analyzed using ANOVA. The difference was statistically significant at P <0.05, P <0.01 or P < 0.01.

Three, result in

1. A fur mouse model for conditional knockout of bone marrow mesenchymal stem cells is constructed by using a Cre-Loxp system, and protein level and mRNA level verification are carried out on mouse gene knockout efficiency, and the result is shown in figure 1.

By utilizing Micro-CT analysis, after the Furin gene is knocked out in bone marrow mesenchymal stem cells, a female or male conditional knockout mouse shows a bone mass increase phenotype, and compared with a wild-type Furin fl/fl mouse, the density of trabeculae of bone tissues of the Furin fl/fl, Prx.1-Cre female or male mouse is obviously increased, the number of trabeculae is obviously increased, the interval of the trabeculae is obviously reduced, and the bone mass is obviously increased (shown in figure 2). This suggests that Furin is a potential target for the treatment of bone mass loss.

On the basis, the bone density of lower limb bones and spinal bones of wild-type Furin fl/fl and conditional knockout Furin fl/fl and Prx.1-Cre mice is analyzed by using an X line, and the results in a figure 3 suggest that the bone density of the lower limb bones and the spinal bones of the conditional knockout mice is obviously higher than that of the wild-type mice.

The significant increase in bone mass in the bone tissue of Furin fl/fl, prx.1-Cre female or male mice compared to Furin fl/fl mice (shown in fig. 4) was shown by bone immunohistochemical staining analysis, suggesting that Furin may be a potential target for the treatment of bone mass loss.

Using hard tissue sectioning techniques, it was further found that Furin fl/fl, prx.1-Cre female or male mice had significantly increased bone mass in bone tissue compared to Furin fl/fl mice (shown in fig. 5).

Therapeutic Effect of CMK on ovariectomized mice

By constructing an OVX bilateral oophorectomy osteoporosis mouse model, in a positive control group, bilateral oophorectomy leads to obvious reduction of bone density of mice, and in vivo intervention is carried out by using CMK (human growth factor kinase), and Micro-CT analysis shows that compared with an OVX model group, bone tissue bone trabecular density of mice in a CMK treatment group is obviously increased, bone trabecular interval is obviously reduced, and bone mass of the mice is obviously increased (shown in figure 6).

Further, by bone immunohistochemical analysis, the CMK-treated group was able to significantly inhibit OVX-induced bone mass loss compared to the OVX model group (shown in fig. 7).

The Micro-CT is consistent with the result of the tissue morphology analysis research, which indicates that the CMK has the function of resisting osteoporosis.

Fourth, discuss

In this study, the inventors observed that CMK can ameliorate bilateral ovariectomy-induced osteoporosis in vivo.

Accordingly, the inventors believe that CMK may have great potential and clinical value for the prevention of bone-related diseases, such as osteoporosis.

More and more studies show that the occurrence and development of osteoporosis are closely related to bone loss caused by imbalance of bone remodeling. Bone remodeling activities require three key cells to be involved in regulation: osteoclasts, osteoblasts and osteocytes. With the aging, the differentiation and proliferation ability and biological functions of osteoblasts are reduced, and osteoclast bone resorption is more active, so that the bone reconstruction activity is unbalanced, the bone mass is lost, and finally osteoporosis is caused.

The precursor protein processing enzyme is involved in the physiological activities of the body, and many signal paths require the precursor protease to shear the precursor so as to exert the biological functions, but the mechanism is not clear. Its family is a family of endoproteases consisting of 9 members: pcsk1, Pcsk2, Furin, Pcsk4, Pcsk5, Pcsk6, Pcsk7, Mbps 1, and Pcsk 9. In previous studies, Furin genes in the precursor protein processing enzyme family are obviously expressed in the osteoblast differentiation process through the statistical analysis of a BioGPS database (http:// BioGPS. gnf. org), however, the research of Furin on the aspects of osteogenic differentiation and bone formation is not clear.

Thus, the inventors generated conditional knockout mice using the hybridization of Furin-Flox and Prx.1-cre mice. Analysis shows that the knockout mice show increased bone mass, which suggests that Furin plays an important role in osteoblast differentiation and function. Therefore, the inventor speculates that the Furin inhibitor CMK has certain influence on bone homeostasis, and can achieve the effect of relieving the bone mass loss diseases, such as osteoporosis.

The inventor simulates an osteoporosis model by constructing bilateral ovariectomy and applies CMK treatment, and finds that the CMK treatment group has obviously increased bone mass compared with a simple OVX operation group, which indicates that CMK has a certain protective effect on osteoporosis.

And (4) conclusion: CMK is used as a specific inhibitor of Furin, has a prospect of being developed into an anti-osteoporosis drug, and provides an effective way for treating osteoporosis. The invention provides a new treatment idea for preventing osteoporosis diseases.

Claims

Use of a Furin inhibitor for the preparation of a medicament for the treatment of osteoporosis.
2. The use of claim 1, wherein the Furin inhibitor is CMK.
3. Use according to claim 1 or 2, wherein the osteoporosis is caused by ovariectomy.